Method for detecting microbe or cell

ABSTRACT

A method for detecting microorganisms or cells wherein microorganisms or cells contained in a sample are captured on the surface of the adhesive layer of a collection sheet having a substrate layer containing a focusing marker, an adhesive layer having a predetermined thickness and deposited on this substrate layer, the microorganisms or cells are stained by a staining reagent, and after being automatically focused, the light receiving optical system or the collection sheet is moved relatively by a distance equivalent to that obtained by adding the distance from the surface of the substrate layer to the position of the focusing marker to the predetermined thickness of the adhesive layer from the focusing position by this autofocusing reference point, the microorganisms or cells on the adhesive layer are automatically focused, and a light is radiated on the focused adhesive layer to measure the image and detect the microorganisms or cells.

TECHNICAL FIELD

The present invention relates to a method of marking with a stainingreagent microorganisms or cells contained in a sample and detecting thesame by measuring their images. The microorganisms mentioned aboveinclude prokaryotes such as microbes, actinomycete and the like,eucaryotes such as yeast and mould, algae, viruses, and so forth. Thecells include cultured cells derived from animals and plants as well aspollens of Japanese cryptomeria and Hinoki. The fields of application ofthe present detection method include medical treatment, manufacture offoodstuffs, water supply and drainage.

BACKGROUND ART

The detection of microorganisms or cells of animals and plants insamples is industrially a very important art for, for example, forconfirming sterilization and detecting any abnormality in the livingcondition of cells.

The method that has been generally used so far to detect bacteria andother microorganisms or cells existing on the surface to be examined andthat cannot be observed by naked eyes is that of measuring by visuallyfixing with naked eyes or by a microscope for actual matters a colonythat emerges by the culture method, or in other words by copying themicroorganisms found on the surface to be examined on an agar mediumrealized in due form with agar by pressing the solid agar medium againstthe surface to be examined and by culturing the microorganisms in asuitable environment on the agar medium as they are. For example, theagar stump method based on the use of FOOD STAMP (made by NissuiPharmaceutical Co., Ltd.) may be mentioned.

The membrane filter method based on the use of a membrane filter and thelike capable of collecting microorganisms is a method of measuring acolony wherein microorganisms are collected while the surface to beexamined are sufficiently wiped with saline, phosphate buffer and thelike and the microorganisms are exposed, and after the microorganismsare collected on the membrane filter as this washed collector isfiltered through a membrane filter, the microorganisms and the liquidculture medium are brought into a sufficiently close contact to form acolony on the filter. The membrane filter method can be used as a methodfor detecting microorganisms without culturing them by having themicroorganisms captured on the filter come in contact with a suitablestaining solution, and the number of colored cells is measured by amicroscope and the like.

However, the agar stump method and the like in which normally an agarstump is used only once on a surface to be examined is inferior in termsof reproducibility due to a variable collection efficiency depending onthe moisture ratio of the agar medium, and sometimes this causesinconveniences in the efficiency of collecting microorganisms. And acommon problem for the culture method is that microorganisms arecontaminated mutually and the impossibility of pure culture due to thereciprocal actions among microorganisms on the medium sometimes causesinconveniences in the subsequent judgments. And the application of theagar medium on the surface to be examined could contaminate the surfaceto be examined. In addition, there is a limitation in that the culturemethod is naturally limited to viable cells and involves a certainnumber of detections omitted. In addition, due to the necessity ofproviding one to two days or more for the period of culture, the culturemethod had an important limitation in that it was impossible to monitormicroorganisms in real time.

And the membrane filter method had a disadvantage in that, although itis possible to filter a liquid subject of examination such as aqueoussolution, non-liquid subjects of examination required tremendous humanefforts to collect microorganisms including sampling by means of anapplicator, the preparation of a washing liquid for exposingmicroorganisms and the like. In addition, the washing and filteringoperation caused the matters collected other than microorganisms toswell and this obstructed the subsequent observations and measurements.

Lately, a microorganism testing method has been proposed to detectpromptly and easily microorganisms on the surface of a solid body,wherein an adhesive layer mainly composed of water-soluble polymer of anadhesive sheet is pressed on the surface to be examined and peeled tocollect the microorganisms found on the surface of the solid body, andthen an aqueous solution containing one or more types of chromogenicsubstance or substances capable of staining microorganisms is made toget into contact with the surface of the adhesive layer, and the numberof stained cells is observed and counted (image measurement) (see forexample Japanese Patent Application Laid Open 10-70976).

However, these are cases of image measurement carried out by using amanually focused microscope or a similar image pickup device, and due toa narrow depth of field when used at a high magnifying power, it oftentakes much time to adjust the focus. Therefore, automatic focusing andautomatic measurement have been desired.

With regard to a method of fluorescent image measurement involving theautomatic focusing mentioned above, an application for the fluorescentimage measurement method invented by some of the inventors of thepresent application was filed by Japanese Patent Application 2002-30648.

FIG. 1 shows an example of device for carrying out the method describedin the Japanese Patent Application 2002-30648. According to the deviceshown in FIG. 1, the fluorescent image measurement method involvingautomatic focusing based on the image information obtained through animage pickup means includes radiating an auto-focusing (AF) light thatilluminates in the wavelength band for fluorescent image measurement bythe light source 2 from the same side as the radiation side ofexcitation beam as shown before irradiating the excitation beam to thesample 1 from the excitation beam source 10, judging the degree offocusing from the image information obtained thereby, driving at leastone of sample 1 or the light receiving system in response to the degreeto search the position of focus, stopping the radiation of the beam forAF upon reaching the position of focus, and then irradiating theexcitation beam to the sample 1 from the light source 10. Thus, afluorescent image measurement can be carried out. This device that usesno penetrating light enables to measure samples collected on the surfaceof the membrane filter.

As the light source 2 for AF, a light-emitting diode or a semiconductorlaser is preferable. The image of samples during the radiation of AFbeam is caught by an image pickup element 7 through an object lens 5, adichroic mirror 3, a filter on the receiving side of fluorescent light 4and an imaging lens 6. As the image pickup element, an element for CCDcamera or an element for CMOS camera is preferable. Images obtained bythe image pickup element 7 are sent to the calculating part 8, where thecontrast is evaluated. For the evaluation of contrast, for example, thedifference of luminance between adjacent pixels is calculated, and thisis carried out by the general AF method wherein the position of themaximum contrast is considered as the position of focusing.

In FIG. 1, 9 represents a stage transfer mechanism, 11 represents acondenser of excitation beam, 12 represents a optical filter, and 13represents a fluorescent light optical filter block. And although notshown in FIG. 1, for the evaluation of the contrast mentioned above, theJapanese Patent Application 2002-30648 discloses the method of markingon the surface of a slide glass for holding the sample, or marking onthe surface of a membrane filter for filtering and collecting the sample(for details, see the Japanese Patent Application 2002-30648). Accordingto the method mentioned above, the adoption of a simple mechanism with alimited number of element enables to avoid the optical quenching of thesample due to the radiation of the excitation beam making it impossibleto detect the same and to autofocus (AF) samples collected on thesurface of the membrane filter and samples with a weak contrast.

In the meanwhile, even the invention described in the Japanese PatentApplication 2002-30648 described above has the following problem.

As the AF mark is provided on the surface of the slide glass for holdingthe sample or on the surface of the membrane filter for filtering andcollecting the sample, the mark provided in the close vicinity ofmicroorganisms and other samples constitutes an optical noise causingthe precision of measurement to fall when an excitation beam is radiatedto carry out a fluorescent image measurement.

In other words, in observing the image of microorganisms or cells, themark on the surface of the membrane filter that holds the sample isreflected on the image, and constitutes a background noise thatobstructs precise measurements. In particular, when observing faintlight emitted by microorganisms or cells, the above-mentioned noise willbe an important problem. For this reason, there has been a demand for amethod of preventing marks on the sample holders (focusing marker) frombeing reflected and suppressing noises.

And the invention described in the Japanese Patent Application2002-30648 mentioned above was mainly concerned with liquid samples andis based on the principle that microorganisms and the like existing onthe surface of solid bodies are sampled with an applicator and the likeas described above and are transformed into samples of liquid into whichthey are dispersed. Therefore, it is impossible to easily monitor inreal time microorganisms existing on the surface of solid bodies andautomatically focus and measure them.

The present invention has been realized by taking into consideration theabove issues, and the object of the present invention is to provide amethod of detecting microorganisms or cells capable of in particulareasily monitoring in real time microorganisms existing on the surface ofsolid bodies and designed to improve the accuracy of automatic focusingand measurements.

DISCLOSURE OF THE INVENTION

In order to resolve the issues mentioned above, the present inventionincludes the following steps in the process of marking microorganisms orcells contained in a sample (including the case of coexistence of bothof them) with a staining reagent and detecting them by image measurement(invention of claim 1 hereof).

1) A step of capturing microorganisms or cells contained in the samplementioned above on the adhesive layer of a collection sheet composed ofa substrate layer having a focusing marker for autofocusing at least onits surface and an adhesive layer having a predetermined thicknessdeposited on the surface of this substrate layer.

2) A step of staining the microorganisms or cells captured as mentionedabove with a staining reagent,

3) A step of autofocusing the focusing marker mentioned above,

4) A step of moving at least one of the light receiving optical systemfor image measurement or the collection sheet relatively by theequivalent distance to the predetermined thickness of the adhesive layerfrom the focusing position by the autofocusing mentioned above as areference point, and focusing on the microorganisms or cells found onthe adhesive layer mentioned above, and

5) A step of radiating light on the surface of the adhesive layer thathad been brought into focus, measuring the images and detectingmicroorganisms or cells.

According to the detection method described above, it is possible toeasily capture microorganisms existing on the surface of solid bodies onthe adhesive layer mentioned above of the collection sheet. And due tothe presence of an adhesive layer between the focusing marker on thesurface of the substrate and microorganisms or cells, the focusingmarker on the surface of the substrate does not create optical noises onthe occasion of image measurements, and clear images of themicroorganisms or cells captured can be obtained and therefore themicroorganisms or cells can be measured with a high precision.Incidentally, the focusing marker on the surface of the substratementioned above may be formed by sandblasting, printing or other surfacetreatments or by creating optical patterns by mixing silica and otherinsoluble grains. The details of these operations will be describedbelow.

The invention according to claim 1 above comprised capturingmicroorganisms or cells on the adhesive layer and then staining them.However, it is possible to stain them in advance of their capture asdescribed below. In other words, the detecting process described inclaim 1 above includes the following steps in place of the steps 1) and2) described above (invention according to claim 2).

1) A step of staining in advance the microorganisms or cells containedin the sample by a staining reagent,

2) A step of capturing the microorganisms or cells contained in thesample that have been stained by the staining reagent in advance on theadhesive layer of a collection sheet composed of a substrate layerhaving at least on its surface a focusing marker for autofocusing and anadhesive layer having a predetermined thickness deposited on the surfaceof this substrate layer.

And in case where the fluorescence observation images of microorganismsor cells are obtained, as an embodiment of the invention according toclaim 1 or claim 2, the invention according to claim 3 described belowis preferable. In other words, in the detecting process according toclaim 1 or 2, the fluorescent reagent mentioned above is chosen as astaining reagent and the excitation light is radiated on the adhesivelayer mentioned above to carry out fluorescent image measurements, andin addition the radiation light for autofocusing on the occasion ofautofocusing on the focusing marker mentioned above will be a light thatincludes a wavelength in the optical wavelength band for the fluorescentimage measurement mentioned above (invention according to claim 3).

The meaning of choosing a light that includes a wavelength of theoptical wavelength band for the fluorescent image measurement mentionedabove as the radiation light for the autofocusing mentioned above isthat it is intended to limit any focusing errors at the time of bringingthe focusing marker into focus and bringing microorganisms or cells intofocus.

In addition, in the detecting process according to any one of claims 1to 3, the adhesive layer mentioned above comprises a non-water solubleadhesive (invention according to claim 4). This arrangement enables,when for example microorganisms or cells are marked with a fluorescentsubstance, to prevent the fluorescent substance from having difficultyin penetrating the adhesive layer, the adhesive layer from dissolvingresulting in a movement of microorganisms or cells that have beencaptured, and the thickness and dimensions of the adhesive layer fromchanging thereby.

Furthermore, in the detecting process according to any one of claims 1to 4 above, the predetermined thickness of the adhesive layer should begreater than the depth of field of the optical system (inventionaccording to claim 5). This enables to measure in such a way that thefocusing marker may not be reflected as the background noise during theobservation of microorganisms or cells.

And as an embodiment different from the invention of the focusing markermentioned above, it is possible to arrange things as described in theinvention according to claim 6 described below. Specifically, in thedetection process according to any one of claims 1 to 5, the focusingmarker should be provided “on the back of or within the substrate layer”in place of “on the surface of the substrate layer”, and “the step ofmoving by a distance equivalent to the value of the predeterminedthickness of the adhesive layer” in the step 4) above should be replacedby “the step of moving by a distance equal to the value of the distanceobtained by adding the value of the distance from the surface of thesubstrate layer to the position of the focusing marker to the value ofthe predetermined thickness of the adhesive layer” (invention accordingto claim 6).

The focusing marker on the back of the substrate should be printed ortreated by other means of surface treatment, and when it is in thesubstrate layer, it may be formed by the creation of an optical patternby mixing silica and other insoluble grains. In the case of theinvention according to claim 6, however, in particular in the opticalpassage from the surface of the adhesive layer to the focusing marker,because of the existence of two different materials of the adhesivelayer and the substrate layer and a relatively greater value of opticaldistance, it is desirable to correct the distance of movement based onthe refractive index of each of different materials as necessary. Andwith regard to the depth of field, all that is required to make thedistance obtained by adding the value of distance from the surface ofthe substrate layer to the position of the focusing marker to the valueof the predetermined thickness of the adhesive layer greater than thevalue of the depth of field of the optical system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration showing an example of configuration of afluorescent image measuring device involving autofocusing described theJapanese Patent Application 2002-30648.

DESCRIPTION OF SYMBOLS

1. Sample

2. Light source for autofocusing

3. Dichroic mirror

4. Optical filter on the receiving side of fluorescent light

5. Objective lens

6. Imaging lens

7. Image pickup element

8. Calculating part

9. Stage moving mechanism

10. Light source for excitation

11. Condenser lens for excitation beam

12. Optical filter

13. Fluorescent filter block

THE BEST MODE FOR CARRYING OUT THE INVENTION

The collection sheet used in the present invention comprises an adhesivelayer composed mainly by a polymer compound deposited on the substratelayer, and includes a focusing marker constituted by insoluble grainswithin or on the surface (including the interface between the substratelayer and the adhesive layer) or on the back of the substrate layer or arelief pattern on the surface of the substrate layer.

As a method of creating a focusing marker on the surface or the back ofthe substrate layer, the method of pressing to bring into relief orcasting at the time of making the film for the substrate layer, themethod of sandblasting or making similar treatments to the surface ofthe substrate layer, the method of printing on the surface of thesubstrate layer and the like can be mentioned. When the surface of thesubstrate layer is brought into relief by extruding the substrate layerat the time of forming the film for the substrate layer, casting,sandblasting and other treatments, the preferable depth of the relief isbetween 0.5 to 20 μm.

As for the printing method of the focusing marker, taking intoconsideration the fact that image contrast is judged at the time offocusing operation, flat all-over printing is not suitable and linear,checkered or dot patterns are preferable. And more preferably, at thetime of obtaining images, a pattern of which at least a borderline isvisible in the field of vision or with a changing color is desirable.

And when a focusing marker is provided within the substrate, the resinconstituting film of the substrate layer may be made by mixing insolublegrains. For the insoluble grains, grains of calcium carbonate, titaniumoxide, carbon black, silica, polystyrene, talc, asbestos, mica, clay,cellulose, starch and the like are indicated, and the grains having anaverage grain diameter of 0.5 to 20 μm are preferably used. Theseinsoluble grains may be substituted by air bubble or carbon dioxidebubble.

Such a focusing marker may be disposed within the substrate layer, onthe surface or on the back of the substrate layer of the collectionsheet, and they may be doubled. For example, mixed insoluble grains ofsilica may be dispersed on the surface of the substrate layer to serveas the focusing marker on the surface of the substrate layer.

The adhesive layer mentioned above is not specially limited, providedthat it has a sufficient power to capture microorganisms or cells on thesurface to be examined and that it is a layer having a level surfacestructure of which the adhesive does not dissolve even if it issubmerged in an aqueous solution for staining microorganisms or cells.However, when microorganisms or cells are marked with a fluorescentsubstance, in order to make it difficult for the fluorescent substanceto penetrate into the adhesive layer, or in order to prevent theadhesive layer from dissolving causing the microorganisms or cellscaptured to move and also to prevent the value of thickness of theadhesive layer from changing, it is preferable to choose a non-watersoluble adhesive for the principal component of the adhesive layer.

As the non-water soluble adhesive, for example acrylic adhesives, rubberadhesives and silicone adhesives may be used. And from the viewpoint ofreducing the impacts on optical characteristics at the time of obtainingfluorescent images, for the substrate layer and the adhesive layer, theadoption of a highly transparent and non-fluorescent acrylic adhesive orsilicone adhesive is preferable.

As acrylic adhesives, those mainly composed of, as monomer, an alkylester of (meth)acrylic acid such as ethyl (meth)acrylate, propyl(meth)acrylate, butyl (meth)acrylate, hexyl (meth)acrylate, octyl(meth)acrylate, 2-ethylhexyl (meth)acrylate, nonyl (meth)acrylate ordecyl (meth)acrylate and, copolymerized therewith, one or morehydrophilic monomers such as (meth)acrylic acid, itaconic acid, maleicacid, hydroxyethyl (meth)acrylate, methoxyethyl (meth)acrylate,ethoxyethyl (meth)acrylate, butoxyethyl (meth)acrylate or ethyleneglycol(meth)acrylate can be mentioned. And it is preferable to crosslink suchan adhesive layer by a treatment with a thermally crosslinking agentsuch as an isocyanate compound, an organic peroxide, an epoxygroup-containing compound or a metal chelate compound or a treatmentwith ultraviolet rays, γ rays, electron beam or the like to enhance itsadhesive characteristics.

As rubber adhesives, it is possible to use those comprising as the mainpolymer natural rubber, polyisobutylene, polyisoprene, polybutene,styrene-isoprene block copolymer, styrene-butadiene block polymer andthe like and, incprporated therewith, a tackifier resin such as a rosinresin, a terpene resin, a coumarone-indene resin, terpene phenolicresin, a petroleum resin and so forth. As for silicone adhesive, anadhesive mainly composed of dimethylpolysiloxane may be exemplified.

From the viewpoint of adhesiveness on the surface to be examined,followability and collectability of microorganisms, it is preferablethat the thickness of such an adhesive layer would be within a range of5 to 100 μm. And at the time of obtaining the fluorescent images of themicroorganisms or cells captured, it is preferable that the smoothness(distance from the top of convex to the bottom of concave) of thesurface of the adhesive layer would be 20 μm or less. When roughness is20 μm or less, the range of focusing of the fluorescent image-acquiringmeans will be extended, which will enable to treat images with a higherprecision. Roughness can be determined by observing the section of theadhesive sheet with a surface roughness meter or an electronicmicroscope and by measuring the difference of altitude from the top ofconvexes to the bottom of concaves of the surface of the adhesive.

The material of the substrate of the collection sheet is notparticularly limited as long as it is non-water soluble, does not formimportant ruggedness on the surface, and is a flexible material whichcan be freely fixed with pressure on a curved surface or a surface ofsample set narrow space. Specifically, sheets, films, fabric, non-wovenfabric, paper composed of polyester, polyethylene, polyurethane,polyvinyl chloride, and polyethylene laminate paper are indicated asexamples. In particular, smooth sheets and films composed of polyester,polyethylene, polyvinyl chloride, polyurethane, etc. are preferable asthe substrate. And the thickness of the substrate is not particularlylimited as long as it is sufficiently strong as a supporting body, but athickness approximately ranging from 5 to 200 μm is preferable.

The collection sheet used in the present invention can be produced bythe methods already known. For example, it is produced by applying asolution containing a macromolecular compound used in the adhesive layeron the substrate and by drying the same at a temperature ranging fromthe room temperature to 200° C. In addition, the calendar method, thecasting method and the extruding method can also be used. When afocusing marker is provided in the substrate, the substrate is producedby carrying out the surface treatment mentioned above or by addinginsoluble grains, and it is preferable to provide the focusing marker onthe substrate before depositing the adhesive layer. The sheet obtainedthus can be cut in a freely chosen form for use.

According to the present invention, it is possible to sterilize thecollection sheet by radiating radiant rays such as electron beams or γrays and bridge at the same time the polymer compounds used in theadhesive layer. And it is also possible to sterilize the collectionsheet by means of ethylene oxide and other similar gases, and tomaintain the sterile condition by enclosing the collection sheet in amicroorganisms insulating packing material in the sterilized condition.

For the purpose of the present invention, the term “microorganisms”includes, as described above, prokaryotes such as microbe andactinomycete, eucaryotes such as yeast and mould, algae, viruses and thelike, and the term “cells” include cultured cells derived from animalsand plants, pollens of Japanese cryptomeria and Hinoki.

According to the detection process of the present invention, it ispossible to stain the microorganisms or cells that will be the subjectof detection with one or more types of chromogenic substance orsubstances. The chromogenic substances are not particularly limited aslong as they develop colors by reacting with the cell componentscontained in the microorganisms that are the subject of examination.However, as respresentative ones, it is possible to mention fluorescentstains that stain nucleic acid and protein. As specific chromogenicsubstances, in the case where microorganisms in general are thesubjects, it is possible to mention fluorescent nucleic acid baseanalogs, fluorescent stain for staining nucleic acid, stain solution forstaining protein, fluorescent probe used in the tissue analysis ofprotein, stain solution used in the analysis of cell membrane andmembrane potential, stain solution used for marking fluorescentantibodies and the like, in the case where aerobic bacteria are thesubjects, stain solutions and the like that develop color by therespiration of cells, in the case where eucaryotes are the subjects, astain solution for staining mitochondria, a stain solution for stainingGolgi bodies, a stain solution for staining endoplasmic reticula, in thecase where the stain solution reacting with intracellular esterase andits modification compounds are directed towards advanced animal cells, astain solution used for the observation of bone tissues, a stainsolution serving as a nerve cell tracer and the like are mentioned, andmicroorganisms or cells stained by these stain solutions can be observedby means of a fluorescent microscope.

By choosing the type of these chromogenic substances, they can beapplied to an extensive field including the measurement of the totalnumber of cells for detecting all the microorganisms, the assay ofstaining and counting only microorganisms having a respiratory activity,the assay of staining and counting only microorganisms having anesterase activity, or the assay of staining and counting specific generaand species of microorganisms by using the double banding methodcombining a plurality of chromogenic substances and so forth.

In the present invention, the microorganisms or cells adhering to thesurface to be examined are efficiently copied and captured by pressingthe collection sheet on the surfaces to be examined including floor,walls, foodstuffs and the like. When the collection sheet is pressed onsurfaces to be examined where there seem to be relatively fewmicroorganisms or cells, a same surface of the collection sheet may bepressed a number of times. As the process of the present inventionrequires no culture as in the case of the agar stump method, there is noneed to worry about the contamination of colonies and any possiblechanges in the phase of cells during their culture, and therefore itenables to capture microorganisms many times. Thus, many microorganismsor cells can be captured by increasing the number of pressing in thesame way as filtering and condensing the microorganisms or cells thatare dispersed in water by the membrane filter method.

Then, the collection sheets on which microorganisms or cells arecollected are cut to a predetermined size as required, and the surfaceon which microorganisms or cells are collected is submerged in anaqueous solution containing a chromogenic substance to stain themicroorganisms or cells. If it is necessary to remove excess chromogenicsubstance, the surface on which the microorganisms or cells arecollected is washed and rinsed with sterilized water. And when it isnecessary to dry the surface where the microorganisms or cells arecollected after they have been stained, it is possible to dry them byair drying, natural drying or depressive desiccation.

Microorganisms or cells are counted and measured by obtaining theiroptical images by an optical microscope having an automatic focusingfunction, a fluorescent microscope, a laser microscope, a laser scanningsite meter, or other suitable optical instruments and by measuring theseimages. The collection sheet of the present invention demonstrates itspower and enables to measure images promptly. In other words, it ispossible to focus the microorganisms or cells captured by focusing onthe focusing marker in the collection sheet by taking advantage of theautomatic focusing function and by shifting the focus by the thicknessfrom the focusing position within the collection sheet to the surface ofthe adhesive layer. As this series of operations does not requirecultivation, the microorganisms found on the adhesive surface of thecollection sheet can be effectively detected within several minutes orten and a few more minutes.

The present invention can be applied, for example, to environmentalresearches for measuring promptly the cleanness of the subjects ofexamination, because the collecting surface can be adhered to thesurface to be examined, the microorganisms found on the surface forexamination are copied, the microorganisms are stained without priorcultivation and the microorganisms can be observed at the single celllevel. And it is possible and practical to capture microorganisms byapplying the collection sheet several times on the surface to beexamined and condense them, because they are captured at the single celllevel. As fields of application, this method can be applied to theenvironmental study of microorganisms at the site of medical treatmentand food processing.

The following is an example of embodiment wherein the fluorescentobservation images of microorganisms or cells fluorescent stained by themethod described above are obtained. Specifically, a reagent fluorescingby the esterase activity of the microorganisms or cells captured by thecollection sheet, for example carboxy fluoresceine diacetate(hereinafter referred to as “CFDA”) is reacted. For obtaining thefluorescent images of the microorganisms or cells fluorescent stained byCFDA, an autofocusing light emitting the fluorescent wavelength light ofCFDA (for example 500-550 nm) is radiated on the collection sheet. Afterfocusing on the marker on the collection sheet, the distance between thedetecting part of the optical system and the sample is moved from thatposition by a distance corresponding to the thickness between thefocusing marker and the surface of the adhesive layer (for example 20μm) and the focus is adjusted to the surface of the adhesive layer. Alight having a wavelength capable of exciting CFDA (for example 450-500nm) is radiated on the focused collection sheet to obtain thefluorescent image of the surface of the adhesive layer. And from thefluorescent images obtained there, microorganisms or cells arerecognized and detected.

EMBODIMENT

The present invention will be described more specifically below byreferring to a plurality of embodiments. However, these are merelyexamples and do not limit in any way the scope of the present invention.

Embodiment 1

1) Fabrication of a Collection Sheet

A copolymer toluene solution with a gel ratio of 40% (w/w) is obtainedby polymerizing isononylacrylate, 2-methoxyethylacrylate and acrylicacid (65/30/5 input weight ratio) using azoisobutyronitrile as apolymerization initiator. This solution is applied on a film of which a25 μm thick transparent polyester non-adhesive surface is scratched to adepth of approximately 1 μm by a #1200 sandpaper so that the thicknessmay be 20 μm when dry and a polyester film 26 μm thick to which powderedsilica with an average grain diameter of 5 μm are mixed, and these filmsare dried for five (5) minutes at 130° C. And then these films aresterilized with γ beam having a dosage of 25 k grey to produce acollection sheet. In the meanwhile, the case of using the powderedsilica as the focusing marker will constitute the embodiment 1-1described further below, and the case of using the treatment of thesurface of the substrate with a sandpaper as the focusing marker willconstitute the embodiment 1-2 described further below.

2) Capture and Staining of Microorganisms

0.1 mL of solution obtained by diluting 100 times with sterilized waterthe culture medium of Staphylococcus epidermidis IFO3762 is filteredthrough a polycarbonate membrane having straight holes with a diameterof 0.4 μm and the microorganisms found on the flat membrane and washedby a sterilized phosphate buffer are taken as the subjects ofexamination, and the collection sheet produced by the step 1) above ispressed on the filter surface and is peeled. Then, a phosphate buffercontaining 0.1% of 6-carboxy fluoresceine diacetate is dripped as astain solution to the surface where the microorganisms are collected.After being left unattended for three (3) minutes at the roomtemperature and stained, the surface where the microorganisms arecaptured is again washed with a phosphate buffer.

3) Counting

The sample images are obtained by means of an optical system providedwith a CCD camera as an image pickup device having a multiplication of10 times. This image information served as the basis of driving as leastone of the mirror barrel of the light receiving system or the samplestage by the personal computer and searching the focusing position. Forthis driving, it is suitable to use a stepping motor capable ofcontrolling positions with a resolving power of approximately 0.5-1 μm.By preparing an optical device having such mechanism (hereinafterreferred to as “the measuring device”), the number of microorganisms iscounted on the surface where microorganisms are collected on thecollection sheet wherein the collected microorganisms are stained.

Specifically, in the first place, at least either one of the opticalsystem tube or the sample stage is moved in the direction of separatingfrom the vicinity of the substrate, and the focus point where thefocusing marker in the form of powdered silica and the like indicatesthe focus image obtained is stored. After further moving over apredetermined distance, for example 20 μm, until the point where thefocusing is completed on the surface of the adhesive layer, the sampleimage is obtained. In the case of fluorescence observation, it ispossible to identify the microorganisms or cells as luminescent spots inthe fluorescent image by radiating an excitation beam having apredetermined wavelength.

And it is possible to measure without causing the focusing marker to bereflected as the background noises at the time of observingmicroorganisms or cells by making the value of the distance between thefocusing marker and the surface of the adhesive layer greater than thedepth of field of the optical system. The depth of field depends on theaperture of the optical system, and in normal microscopic observation,it is several μm. As a result, it is possible to prevent the focusingmarker from being reflected on the image obtained and constituting thebackground noises by setting the distance between the focusing markerand the surface of the adhesive layer at 20 μm.

The number of microorganisms or cells found in the field of vision canbe counted by measuring the image obtained. And it is possible to reducestatistical variation and to measure more accurately by driving eitherone of the mirror barrel or the sample stage, observing differentpositions on the sample and counting the number of microorganisms orcells in a plurality of fields of vision. In the present embodiment, thesample stage was driven, the images of a total of 70 fields of visionwere obtained and the number of bacteria contained there was counted.And a sterilized solution was chosen as the subject of examination inplace of a diluted culture broth, and the adhesive surfaces ofcollection sheets on which microorganisms are not captured were alsomeasured in the same way.

4) Data Analysis

The same samples as those used in the measurement mentioned above weremeasured by the culture method to compare with the measured value ofnumber of bacteria by the present invention. The number of bacteriameasured by the culture method totaled 3,028/mm². The measurement resultof the culture method was compared with the measurement value for thenumber of cells based on the present invention that served as thereference, in other words by the recovery ratio of bacteria.

And the same value was compared with the case of no focusing markerbeing used (Comparative Example 1) described below.

Comparative Example 1

The collection sheet was produced in the same way as the Embodiment 1except that a transparent polyester film 25 μm thick of which notreatment was made on the substrate, and microorganisms were captured,stained and washed.

Both the measurement result of Embodiments and the Comparative Example 1are shown in Table 1. Incidentally, in the remark column of Table 1, asstated above, the case of using powdered silica as the focusing markeris shown as the Embodiment 1-1, the case of using the sandpapertreatment of the substrate surface as the focusing marker is shown asthe Embodiment 1-2, and the cases marked with a suffix “a” show caseswhere no microorganism was available for examination. The same thingapplies to the Comparative Example 1. TABLE 1 Number of RecoveryFocusing Microorganisms bacteria ratio of marker examined measuredbacteria Remarks Powdered S. epidermidis 3,149/mm²   104%  Embodimentsilica 1-1 (in the substrate) Powdered None 29/mm² 1% Embodiment silica1-1a (in the substrate) Papersand S. epidermidis 2,846/mm²   94% Embodiment treatment 1-2 on the substrate Papersand None 12/mm² <1%  Embodiment treatment 1-2a on the substrate None S. epidermidis  0/mm² 0%Comparative Example 1 None None Unmeasurable — Comparative (no focusing)Example 1a

As Table 1 shows clearly, the automatic focusing function worked on thefocusing marker of the collection sheet and S. epidermidis could bemeasured in the Embodiment 1-1 and the Embodiment 1-2. The reason whythe collection sheets that do not capture at all microorganisms(Embodiment 1-1a and Embodiment 1-2a) detect a small number ofmicroorganisms is that the microorganisms and fluorescent grain noisesin the environment of measurement may have slipped in and that it may beaffected by mistake in the image processing. The measurements of3,149/mm² and 2,846/mm² in Table 1 seem to include errors of a similarmagnitude.

In the case of Comparative Example 1 in which no focusing marker wasprovided, no focusing was completed and no measurement could be carriedout. Even if no focusing marker is available, the samples themselves(for example, S. epidermidis captured) are often mistaken as apseudo-focusing marker, which can be subjects of automatic focusing. Insuch a case, however, a sample image is obtained at a position forciblyshifted further from the focusing position by a predetermined distance(for example, 20 μm), microorganisms cannot be accurately focused andthe luminance spots derived from the microorganisms cannot be identifiedin the image. When no focusing marker is provided in the collectionsheet like this, no appropriate focusing can be made on samples, andtherefore it has become clear that it is incomplete as a measurementsystem.

Embodiment 2

A procedure similar to that of the Embodiment 1 was considered exceptthat the microorganism to be tested is Escherichia coli K-12 and that acollection sheet including a substrate in which silica is mixed isadopted. The results will be shown in Table 2 along with the ComparativeExample 2.

(Control 2)

The collection sheet was produced in the same way as the Embodiment 2except that a transparent polyester film 25 μm thick of which notreatment was made on the substrate, and microorganisms were captured,stained and washed. TABLE 2 Micro- Number of Recovery Focusing organismsbacteria ratio of marker examined measured bacteria Remarks Powdered E.coli 2,147/mm² 62% Embodiment 2 silica K-12 (in the substrate) None E.coli   0/mm²  0% Comparative K-12 Example 2

In the Embodiment 2, the automatic focusing function works on thefocusing marker of the collection sheet, and the number of E. coli K-12bacteria could be measured. However, the recovery ratio of bacteriavaries depending on the type of bacteria due to differences instainability by the reagent (in the Embodiment 2 above, 6-carboxyfluoresceine diacetate) in addition to the impact of the samplecondition and depending on the microorganism. In the case of S.epidermidis of the Embodiment 1 mentioned above, it had a value close toapproximately 100%. However, in the case of E. coli K-12 of theEmbodiment 2 and the case of E. coli O157 described below, it wasapproximately 60%. In this case, the measurement value of the presentinvention can be converted by the recovery rate of bacteria to be thereal value.

In the case of the Comparative Example 2 without any focusing marker,focusing could not be completed and no measurement could be carried out.Therefore, when no focusing marker is provided in the collection sheet,the whole system is unsuitable as a measuring system because it isimpossible to focus.

Embodiment 3

The microorganism E. coli O157 was chosen as the microorganism to betested, and this embodiment was examined in the same way as theEmbodiment 2. However, with regard to staining, FITC labeled antibody E.coli O157 antibody (made by KPL Inc., diluted by phosphate buffer salineso as to be 0.05 mg/ml) was chosen for the bacteria, and after five (5)minutes of staining, the bacteria were washed with sterilized water. Theresults are shown in Table 3 along with the following ComparativeExample 3.

Comparative Example 3

The collection sheet was produced in the same way as the Embodiment 3except that a transparent polyester film 25 μm thick of which notreatment was made on the substrate, and microorganisms were captured,stained and washed. TABLE 3 Micro- Number of Recovery Focusing organismsbacteria ratio of marker examined measured bacteria Remarks Powdered E.coli 2,186/mm² 60% Embodiment 3 silica O157 (in the substrate) None E.coli   0/mm²  0% Comparative O157 Example 3

In the Embodiment 3, the automatic focusing function works on thefocusing marker of the colleting sheet, and the number of E. coli O157bacteria could be measured. Although the staining mechanism ofmicroorganisms is different from that of the Embodiment 1 and theEmbodiment 2, there was absolutely no inconvenience in detection.

In the case of the Comparative Example 3 without any focusing marker, nofocusing could be carried out and it was impossible to measure.

Embodiment 4

The culture broth E. coli K-12 was stained, measured, and the timerequired for counting a freely chosen number of bacteria was measuredaccording to the method described in the Embodiment 2. The results areshown in Table 4 along with the Comparative Example 4 shown below.

Comparative Example 4

The culture broth E. coli K-12 was diluted as required by a phosphatebuffer and 6-carboxy fluoresceine diacetate was added thereto so thatits final density may be 0.1%, and a staining was carried out for three(3) minutes at the room temperature. This solution was filtered througha polycarbonate membrane to collect the bacteria. The membrane thatcollected the bacteria was observed by a fluorescent microscope under ablue excitation beam at a multiplication of 400 times, and the number offluorescent cells was counted. TABLE 4 Micro- Number of Measuringorganism bacteria Time required method tested counted for countingRemarks Present E. coli 20,000 10 minutes Embodiment 4 invention K-12 ormore Observation E. coli About 45 minutes Comparative by fluorescentK-12 3,000 Example 4 microscope, visual counting

The detection method of the present invention required only 10 minutesto analyze 20,000 or more bacteria.

The method used in the Comparative Example 4, on the other hand,required 45 minutes to count approximately 3,000 bacteria. This is notonly attributable to the trouble of counting by man power but also tothe necessity of changing the field of vision of the fluorescentmicroscope in the process of counting and moreover to the time requiredfor the operation of focusing each time.

The results shown in Table 4 demonstrates that the method of the presentinvention is effective for promptly and easily detecting microorganismsor cells.

INDUSTRIAL APPLICABILITY

As described above, according to the present invention, themicroorganisms or cells contained in samples are captured on the surfaceof the adhesive layer of a collection sheet comprising a substrate layercontaining on the surface, the back or within the substrate a focusingmarker for autofocusing, an adhesive layer having a predeterminedthickness and deposited on the surface of this substrate layer, themicroorganisms or cells are stained by a staining reagent before orafter their capture, and after being automatically brought into focus bythe focusing marker, at least either one of the light receiving opticalsystem for image measurement or the collection sheet is moved relativelyby a distance equivalent to the distance obtained by adding the value ofdistance from the surface of the substrate layer to the position of thefocusing marker to the value of the predetermined thickness of theadhesive layer (if the focusing marker is provided on the surface of thesubstrate layer, the added value is zero) from the focusing position bythis autofocusing as the reference point, the microorganisms or cells onthe adhesive layer are brought into focus, and a light is radiated onthe surface of the focused adhesive layer to image measure and detectthe microorganisms or cells. Therefore, the present invention enables inparticular to monitor easily and in real time microorganisms existing onthe surface of solid bodies, and in addition provides a method ofdetecting microorganisms or cells with an improved accuracy of measuringautomatic focusing.

1. A method of marking by a staining agent microorganisms or cellscontained in a sample and detecting the same by measuring image,comprising; 1) a step of capturing said microorganisms or cellscontained in said sample on the adhesive layer of a collection sheetcomposed of a substrate layer having a focusing marker for autofocusingat least on its surface and an adhesive layer having a predeterminedthickness deposited on the surface of this substrate layer, 2) A step ofstaining said captured microorganisms or cells by a staining reagent, 3)A step of autofocusing said focusing marker 4) A step of moving at leastone of the light receiving optical system for image measurement or thecollection sheet relatively by the equivalent distance to the value ofsaid predetermined thickness of the adhesive layer from the focusingposition by said autofocusing as a reference point to bring saidmicroorganisms or cells on the adhesive layer into focus, and 5) A stepof radiating light on the surface of said adhesive layer that had beenbrought into focus and detecting the microorganisms or cells bymeasuring image.
 2. The method of detecting microorganisms or cellsaccording to claim 1 comprising the following steps in place of saidsteps 1) and 2). 1) A step of staining said microorganisms or cellscontained in said sample in advance by a staining reagent, and 2) A stepof capturing said microorganisms or cells contained in the samplestained in advance by a staining reagent on said adhesive layer of acollection sheet comprising a substrate layer having a focusing markerfor autofocusing at least on its surface and an adhesive layer having apredetermined thickness deposited on the surface of this substratelayer.
 3. The method of detecting microorganisms or cells according toclaim 1, wherein said staining reagent is a fluorescent reagent, anexcitation light is radiated onto the surface of said adhesive layer tomeasure fluorescent image, and the radiation light for autofocusing whensaid focusing marker is automatically brought into focus is a light thatincludes a wavelength of the optical wavelength band for saidfluorescent light image measuring.
 4. The method of detectingmicroorganisms or cells according to claim 1, wherein said adhesivelayer comprises a non-water soluble adhesive.
 5. The method of detectingmicroorganisms or cells according to claim 1, wherein the value of thepredetermined thickness of said adhesive layer is greater than the depthof field of the optical system.
 6. The method of detectingmicroorganisms or cells according to claim 1, wherein said focusingmarker is provided “on the back of the substrate layer or within thesubstrate layer” in place of said “on the surface of the substratelayer,” and “moving by a distance equivalent to the distance obtained byadding the value of distance from the surface of the substrate layer tothe position of the focusing marker to the value of the predeterminedthickness of the adhesive layer” in place of “moving by an equivalentdistance to the value of said predetermined thickness of the adhesivelayer” in said step 4).
 7. The method of detecting microorganisms orcells according to claim 2, wherein said staining reagent is afluorescent reagent, an excitation light is radiated onto the surface ofsaid adhesive layer to measure fluorescent image, and the radiationlight for autofocusing when said focusing marker is automaticallybrought into focus is a light that includes a wavelength of the opticalwavelength band for said fluorescent light image measuring.